Development of the limbs and external genitalia require the coordination of many different regulatory pathways and signaling events. While it is now clear that important components of the limb and genital appendage gene networks are shared, remarkably little is known about the cis- regulatory elements that contribute the transcriptional control of these networks. The Tbx4, Pitx1, and Isl1 transcription factors are among a very small number of hind limb-specific genes that function in the hind limb but not the forelimb. Though work in animal models and humans has shown a central role for these factors in leg formation, we have almost no knowledge of the functional interactions these transcription factors make with the network of cis-regulatory elements that promote limb development. In addition, our preliminary data indicates that at least of one these factors, Tbx4, is also an important regulator of genital growth, suggesting that these genes may exhibit overlapping functional interactions with appendage cis-regulatory elements active in the hind limbs and genitals. The objective of this proposal is to determine how Pitx1, Tbx4, and Isl1 influence the development of appendages, by investigating their interactions with the suite of cis- regulatory elements active in the developing hind limbs and genitalia. This objective will be accomplished by 1) identifying the set of enhancer elements that are bound and directly regulated by Pitx1 in the hind limbs and genitals, 2) determining whether Tbx4 directs the outgrowth of the legs and external genitalia via similar mechanisms, and 3) establishing whether the initiation of hind limb bud and genital bud formation is regulated by Isl1 through a common set of regulatory interactions. The knowledge gained from these studies will help to achieve our long-term goal of understanding how the disruption of specific regulatory interactions can lead to malformations of the limbs and genitals in humans.